Abstract
We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.
Highlights
We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy
To test if the immunotherapy-resistant CD19-105R and Δex2 variants can be tagged with alternative epitopes for flow cytometry, we generated multiple retroviral constructs expressing full-length CD19 (FL-CD19) and bearing hemagglutinin (HA), FLAG, vesicular stomatitis virus G protein (VSVg), and His tags
While in some epitope-negative relapses CD19 expression is extinguished at the level of dedifferentiation into myeloid lineages [9, 10], in our previous paper we described a novel mechanism of resistance to CD19-directed immunotherapy that involved alternative splicing of CD19 exon 2 [11, 34]
Summary
We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy It was based on in-frame insertions in or skipping of CD19 exon 2. While in some sCD19-negative relapses CD19 expression is extinguished at the level of dedifferentiation into myeloid lineages [9, 10], we recently described a posttranscriptional mechanism of acquired resistance to CART-19 compatible with the B-cell phenotype (11; reviewed in references 12 and 13) It is based on the selective loss of CD19 extracellular and transmembrane domains, primarily through skipping of exon 2, which encodes the first of the two constant region 2 (C2)-type Ig-like loops [14]. We describe the nature of sCD19 loss resulting from mutations that do not affect alternative splicing
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